Reactive astrogliosis and the subsequent formation of a glial scar are robust phenomena that occur following diverse CNS injuries. Surprisingly, the molecular signals that control the proliferation of reactive astrocytes or their functions in vivo are poorly understood. Defining the molecular control of reactive astrocyte proliferation and function may lead to therapeutic strategies that modify specific signals in reactive astrocytes to preserve tissue or improve recovery after CNS injury. We demonstrate that intra-arterial infusion of the gamma secretase (GS) inhibitor DBZ (Dibenzazepine) after stroke significantly reduced the proliferation of reactive astrocytes in the peri-infarct area of the cortex, significantly reduced the expression of glial fibrillary acidic protein (GFAP, a marker of activated hypertrophic astrocytes), and significantly increased stroke infarct volumes. The absence of reactive astrocytes after stroke and DBZ treatment correlated with a significant increase in the number of CD45-positive inflammatory cells that invaded the stroke penumbra. Similarly, stereotaxic injection of DBZ directly into the cortex reduced the numbers of proliferating reactive astrocytes surrounding the brain stab injury (needle track) compared with vehicle-injected controls. Reactive astrocytes surrounding the brain stab injury that remained after DBZ injection possessed an altered morphology with a significant reduction in average number of processes, number of branch points, and number of branch ends. Immunohistochemistry with antisera specific to GS cleavage products demonstrated nuclear localization of NICD1 (Notch1) and AICD in reactive astrocytes after cortical injury. DBZ blocks the catalytic activity of Presenilin 1, a component of GS. Experiments designed to specifically delete Notch1 and APP from reactive astrocytes prior to stroke using conditional knockout mice demonstrated that both regulate cortical reactive astrocytes in the peri-infarct area after stroke. Collectively our results indicate that Presenilin 1, Notch1, and APP regulate reactive astrocytes after stroke. Specific Aims: 1. To determine if Presenilin 1 acts as a global regulator of reactive astrogliosis after stroke. 2. To determine whether Notch1 or APP signaling controls the proliferation, morphology, and/or anti-inflammatory functions of reactive astrocytes after stroke. 3. To determine whether Presenilin 1, Notch1, or APP expression in reactive astrocytes is necessary for repair of the blood brain barrier after stroke. PUBLIC HEALTH RELEVANCE: Reactive astrogliosis and the subsequent formation of a glial scar are robust phenomena that occur following diverse CNS injuries. Surprisingly, the molecular signals that control the proliferation, morphology, and functions of reactive astrocytes in vivo are poorly understood. Defining the molecular control of reactive astrocytes may lead to therapeutic strategies that modify specific signals in reactive astrocytes to preserve tissue or improve recovery after CNS injuries such as stroke.